ANGPTL3 Antibody

What is ANGPTL3 and how does it affect lipid metabolism?

ANGPTL3 (Angiopoietin-like protein 3) is a 460-amino-acid polypeptide primarily expressed in the liver that plays a crucial role in lipid metabolism. It functions as an inhibitor of lipoprotein lipase (LPL) and endothelial lipase (EL), enzymes responsible for hydrolyzing triglycerides (TG) and phospholipids in plasma lipoproteins .

ANGPTL3 inhibits LPL activity through a specific mechanism: it induces conformational changes in LPL that increase its susceptibility to cleavage by proprotein convertases, promotes dissociation of LPL from cell surfaces, and directly inhibits LPL's catalytic activity . Additionally, ANGPTL3 inhibits EL, which primarily hydrolyzes HDL phospholipids . This dual inhibitory action on both LPL and EL explains why targeting ANGPTL3 affects multiple lipid parameters simultaneously.

Genetic studies in humans have demonstrated that individuals with loss-of-function mutations in both ANGPTL3 alleles exhibit pan-hypolipidemia characterized by reduced plasma TG, LDL-C, and HDL-C levels, along with increased plasma LPL activity . This genetic evidence strongly supports ANGPTL3 as a therapeutic target for managing hyperlipidemia.

What is the molecular structure and binding mechanism of anti-ANGPTL3 antibodies?

Anti-ANGPTL3 antibodies are typically fully human monoclonal antibodies designed to bind with high specificity and affinity to the ANGPTL3 protein. These antibodies recognize specific epitopes on the ANGPTL3 structure that are critical for its interaction with LPL and/or EL.

For example, the monoclonal antibody REGN1500 binds ANGPTL3 from human, mouse, rat, and monkey with comparable high affinities (KD = 0.26–1.28 nM) . Surface plasmon resonance studies have confirmed that these antibodies do not cross-react with other members of the angiopoietin-like protein family, such as ANGPTL4, ANGPTL5, or ANGPTL8 .

The binding mechanism typically involves the antibody recognizing specific domains on ANGPTL3 that are required for its inhibitory action on lipases. For instance, anti-ANGPTL3/8 antibodies target a leucine zipper-like motif within the ANGPTL3/8 complex that represents the LPL-inhibitory region . This binding effectively blocks the interaction between ANGPTL3/8 and LPL, preventing the inhibition of LPL activity.

How are ANGPTL3 antibodies developed and characterized in the laboratory?

The development of ANGPTL3 antibodies follows a systematic process involving multiple stages of immunization, selection, and characterization:

• Immunization: Transgenic mice expressing human immunoglobulin variable regions (e.g., AlivaMab) are immunized with recombinant human ANGPTL3 or ANGPTL3/8 complex using standard procedures .

• B-cell isolation: Three to five days after the final immunization boost, lymph nodes and/or spleens are harvested to generate single-cell suspensions. Antigen-specific B-cells are then enriched through cell sorting using biotinylated or fluorophore-labeled ANGPTL3/8 .

• Variable region cloning: The variable regions from selected B-cells are cloned following single-cell PCR amplification .

• Initial screening: Antibodies are first screened using an anti-ANGPTL3/8 capture ELISA. The IgG is captured with an anti-human kappa antibody and then tested for binding to biotin-labeled antigen, which is detected using alkaline phosphatase–labeled neutravidin .

• Specificity characterization: Bio-layer interferometry or surface plasmon resonance is used to assess binding specificity. For example, anti-ANGPTL3/8 antibodies are evaluated for binding to the ANGPTL3/8 complex versus free ANGPTL3 or ANGPTL8 .

• Affinity determination: Kinetic analyses determine the equilibrium dissociation constant (KD) from the ratio of the dissociation rate constant to the association rate constant (KD = kd/ka) .

• Functional testing: In vitro assays evaluate the antibody's ability to block ANGPTL3-mediated inhibition of LPL. IC50 values typically range from 1.0 to 13.6 nM for effective antibodies .

What are the structural determinants of ANGPTL3/8 that facilitate antibody targeting?

Research has identified a critical leucine zipper-like motif within the ANGPTL3/8 complex that represents a key structural determinant for antibody targeting. This motif is located within the anti-ANGPTL3/8 epitope, the LPL-inhibitory region, and the ApoA5-interacting region . This structural insight suggests that ApoA5 may lower triglycerides by competing with LPL for the same ANGPTL3/8-binding site.

To characterize these structural determinants, researchers have employed advanced techniques including:

• Hydrogen-deuterium exchange mass spectrometry (HDXMS): This technique allows precise mapping of the interaction sites between ANGPTL3/8 and its binding partners, including LPL, ApoA5, and anti-ANGPTL3/8 antibodies .

• Molecular modeling: Computational approaches help predict and visualize the three-dimensional structure of the ANGPTL3/8 complex and its interactions with antibodies .

• Transmission electron microscopy (TEM): This imaging technique provides visualization of the ANGPTL3/8 complex and its structural changes upon antibody binding .

For effective antibody development, targeting the leucine zipper-containing epitope recognized by both LPL and ApoA5 appears to be crucial for maximally decreasing triglycerides by suppressing ANGPTL3/8-mediated LPL inhibition .

How do different anti-ANGPTL3 antibodies compare in terms of binding affinity and functional inhibition?

Several anti-ANGPTL3 antibodies have been developed, with variations in their binding properties and functional outcomes. Here is a comparative analysis based on available data:

The functional inhibition of these antibodies extends beyond mere binding, as demonstrated by in vitro LPL activity assays. REGN1500 effectively blocks the inhibition of LPL by ANGPTL3 at concentrations within 2.5-fold of the EC50 value for each species tested . Similarly, the anti-ANGPTL3/8 antibody potently blocks ANGPTL3/8-mediated LPL inhibition in vitro .

These differences in binding properties and functional outcomes may translate to varying efficacy in reducing plasma lipids in vivo, as observed in different animal models and clinical studies.

What molecular mechanisms explain the differential effects of ANGPTL3 antibodies on different lipid parameters?

ANGPTL3 antibodies affect multiple lipid parameters through complex molecular mechanisms:

• Triglyceride reduction: Anti-ANGPTL3 antibodies block ANGPTL3's inhibitory effect on LPL, enhancing the hydrolysis of triglycerides in triglyceride-rich lipoproteins. In mouse models, REGN1500 increases LPL activity and decreases plasma TG levels by ≥50% .

• LDL-C reduction: The mechanism of LDL-C reduction is less straightforward but may involve increased LDL receptor availability, enhanced LDL clearance, and reduced VLDL production in the liver. In clinical studies, SHR-1918 demonstrated dose-dependent LDL-C reductions of 21.7%, 27.3%, and 29.9% with 150, 300, and 600 mg every 4 weeks, respectively .

• HDL-C reduction: Studies in EL knockout mice revealed that anti-ANGPTL3 antibodies like REGN1500 reduce serum HDL-C through an EL-dependent mechanism . By blocking ANGPTL3's inhibition of EL, these antibodies enhance EL-mediated HDL phospholipid hydrolysis, leading to accelerated HDL catabolism.

• Apolipoprotein effects: Anti-ANGPTL3 antibodies also reduce apolipoprotein B levels, further contributing to their lipid-lowering effects .

The pan-lipid-lowering effect of ANGPTL3 inhibition mirrors the lipid profile observed in individuals with loss-of-function mutations in ANGPTL3, supporting the concept of genetic validation for this therapeutic approach .

What are the optimal experimental designs for evaluating ANGPTL3 antibody efficacy in preclinical models?

Optimal experimental designs for evaluating ANGPTL3 antibody efficacy in preclinical models should incorporate multiple components:

• Selection of appropriate animal models:

  • Normolipidemic models (e.g., C57Bl/6 mice) to assess basic lipid-lowering effects

  • Dyslipidemic models to mimic human disease states

  • Humanized models expressing human ANGPTL3 to better predict clinical efficacy

  • Non-human primates (e.g., cynomolgus monkeys) for translational studies

• Study duration:

  • Acute studies (single dose) to assess immediate pharmacodynamic effects

  • Chronic administration (e.g., 8 weeks) to evaluate sustained efficacy and potential adaptive responses

• Dosing regimens:

  • Dose-response studies to establish the minimal effective dose

  • Different administration frequencies (e.g., every 4 weeks vs. every 8 weeks) to determine optimal dosing interval

• Comprehensive lipid profiling:

  • Measure multiple lipid parameters (TG, LDL-C, HDL-C, non-HDL-C)

  • Quantify apolipoproteins (ApoB, ApoA5, etc.)

  • Analyze lipoprotein subfractions

• Mechanistic assessments:

  • LPL activity assays in post-heparin plasma

  • Tissue lipid content analysis (liver, adipose, heart)

  • Lipoprotein kinetic studies

For example, the evaluation of REGN1500 included administration to normolipidemic C57Bl/6 mice to assess acute effects on LPL activity and plasma TG, followed by chronic administration to dyslipidemic mice for 8 weeks to evaluate effects on multiple lipid parameters and tissue lipid contents . Similarly, studies in cynomolgus monkeys assessed the antibody's efficacy in a species more closely related to humans, especially in animals with severe hypertriglyceridemia (TG > 400 mg/dl) .

What techniques are used to measure antibody binding and inhibition of ANGPTL3 in vitro?

Several sophisticated techniques are employed to measure antibody binding and inhibition of ANGPTL3 in vitro:

• Surface Plasmon Resonance (SPR):

  • Used to determine binding kinetics (association and dissociation rates)

  • Calculates equilibrium dissociation constant (KD)

  • Assesses binding specificity to ANGPTL3 versus related proteins

  • Example protocol: Recombinant ANGPTL3 proteins are immobilized on sensor chips via anti-histidine capture, and antibodies at various concentrations (e.g., 0.39-50 nM) are injected across the chip surface

• Bio-layer Interferometry:

  • Alternative technique for measuring binding kinetics

  • Used to study interactions between ANGPTL3/8 and LPL complexed with GPIHBP1

  • Can assess competition between antibodies and natural binding partners

  • Example: ANGPTL3/8 antibodies are immobilized on streptavidin biosensors and incubated with ANGPTL3, ANGPTL8, or ANGPTL3/8 complex (5 μg/ml)

• ELISA-based Methods:

  • Anti-ANGPTL3/8 capture ELISA for initial screening

  • IgG captured with anti-human kappa antibody and tested for binding to biotin-labeled antigen

  • Detection via alkaline phosphatase–labeled neutravidin

• LPL Activity Assays:

  • Functional assays measuring LPL-mediated hydrolysis of triglyceride substrates

  • Assesses antibody's ability to reverse ANGPTL3-induced inhibition of LPL

  • Determines IC50 values for functional inhibition

  • Results reported as percentage of control LPL activity

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDXMS):

  • Maps exact binding sites on ANGPTL3/8 complex

  • Identifies structural changes upon antibody binding

  • Provides insights into molecular mechanisms of inhibition

These techniques together provide comprehensive characterization of antibody binding properties and functional effects on ANGPTL3's inhibitory activity.

How can researchers optimize ANGPTL3 antibody production and purification for research applications?

Optimizing ANGPTL3 antibody production and purification for research applications involves several critical steps:

• Immunization Strategy:

  • Use transgenic mice expressing human immunoglobulin variable regions (e.g., AlivaMab)

  • Immunize with recombinant human ANGPTL3/8 complex using standard procedures

  • Employ multiple immunization boosts followed by a final non-adjuvant boost

• B-cell Selection:

  • Harvest lymph nodes and/or spleens 3-5 days after final boost

  • Generate single-cell suspensions

  • Enrich antigen-specific B-cells using biotinylated or fluorophore-labeled ANGPTL3/8

  • Deplete ANGPTL3-positive cells to enhance specificity for the ANGPTL3/8 complex

• Variable Region Cloning:

  • Perform single-cell PCR amplification of variable regions

  • Clone variable regions into expression vectors

  • Verify sequence integrity

• Expression System Selection:

  • Use mammalian expression systems (e.g., CHO or HEK293 cells) to ensure proper folding and post-translational modifications

  • Optimize culture conditions for high antibody yields

  • Consider stable cell line development for consistent production

• Purification Strategy:

  • Implement a multi-step purification process:
    a. Protein A or G affinity chromatography for initial capture
    b. Ion exchange chromatography for charge variant separation
    c. Size exclusion chromatography for aggregates removal

  • Perform thorough quality control testing:
    a. SDS-PAGE and Western blotting
    b. Endotoxin testing
    c. Binding affinity assessment
    d. Functional activity testing

• Antibody Formatting Options:

  • Consider different antibody formats based on research needs:
    a. Full IgG for standard applications
    b. Fab fragments for applications requiring smaller size
    c. Bispecific formats for dual targeting
    d. Tagged versions for detection or purification purposes

By following these optimized procedures, researchers can produce high-quality anti-ANGPTL3 antibodies suitable for various research applications, from basic mechanistic studies to preclinical efficacy assessment.

What clinical trial designs have been most informative for evaluating ANGPTL3 antibodies in humans?

Clinical trial designs for ANGPTL3 antibodies have evolved to address specific research questions and patient populations. The most informative designs include:

• Phase 2 Dose-Finding Studies:

  • Multicenter, randomized, double-blind, placebo-controlled design

  • Sequential dose-escalation approach

  • Randomization ratios favoring active treatment (e.g., 4:1 active/placebo)

  • Multiple dosing regimens (e.g., Q4W vs. Q8W)

  • Treatment periods of sufficient duration (e.g., 16 weeks) to assess efficacy and safety

  • Example: SHR-1918 was evaluated in 333 patients enrolled sequentially into 8 dose cohorts, receiving 150, 300, or 600 mg every 4 weeks, or 600 mg every 8 weeks

• Target Population Selection:

  • Patients with suboptimally controlled hyperlipidemia despite standard lipid-lowering therapies

  • Moderate or higher risk of atherosclerotic cardiovascular disease

  • Run-in periods (4-8 weeks) on standard lipid-lowering therapies to establish baseline lipid levels

  • Specific genetic hyperlipidemia populations (e.g., homozygous familial hypercholesterolemia for evinacumab)

• Endpoint Selection:

  • Primary endpoints focused on percentage change from baseline in key lipid parameters (LDL-C, TG)

  • Secondary endpoints including non-HDL-C, apolipoprotein B, and other lipid markers

  • Safety assessments through laboratory tests and monitoring adverse events

• Extension Studies:

  • Open-label extension phases to assess longer-term efficacy and safety

  • Varied treatment durations (e.g., 36-40 weeks) to evaluate sustained effects

  • Opportunity to test different dosing regimens in the same patient population

These clinical trial designs have provided robust data on the dose-response relationships, optimal dosing frequencies, target patient populations, and safety profiles of ANGPTL3 antibodies, informing their potential place in lipid management strategies.

How do ANGPTL3 antibodies perform in patients with different types of dyslipidemia?

ANGPTL3 antibodies demonstrate varying efficacy across different types of dyslipidemia, reflecting their unique mechanism of action:

• Hypertriglyceridemia:

  • ANGPTL3 antibodies show robust triglyceride-lowering effects

  • Particularly effective in severe hypertriglyceridemia

  • Can normalize plasma TG levels even in subjects with baseline TG > 400 mg/dl

  • Potential application in familial chylomicronemia syndrome (FCS)

• Hypercholesterolemia:

  • Substantial LDL-C reductions observed across studies

  • SHR-1918 demonstrated dose-dependent LDL-C reductions of 21.7-29.9% at doses of 150-600 mg Q4W

  • Evinacumab reduced LDL-C by approximately 50% in patients with homozygous familial hypercholesterolemia

  • Particularly valuable in patients with inadequate response to standard lipid-lowering therapies

• Mixed Dyslipidemia:

  • Simultaneously reduces multiple lipid parameters (TG, LDL-C, non-HDL-C, apoB)

  • SHR-1918 reduced both LDL-C and TG in patients with suboptimally controlled hyperlipidemia

  • Addresses the complex lipid abnormalities often seen in patients with metabolic syndrome or diabetes

• HDL-C Effects:

  • Unlike other lipid-lowering therapies, ANGPTL3 inhibition typically reduces HDL-C levels

  • This effect is mediated through increased EL activity

  • The clinical implications of HDL-C reduction in this context are still being evaluated

The pan-lipid-lowering effect of ANGPTL3 antibodies makes them particularly promising for patients with complex dyslipidemia patterns or those who have not achieved treatment goals with standard therapies. Their efficacy profile mimics the lipid pattern observed in individuals with natural loss-of-function mutations in ANGPTL3, supporting the concept of genetic validation for this therapeutic approach .

What are the most significant challenges in translating ANGPTL3 antibody research to clinical practice?

Despite promising results, several significant challenges exist in translating ANGPTL3 antibody research to widespread clinical practice:

• Long-term Safety Considerations:

  • Limited long-term safety data beyond phase 2/3 trials

  • Need to monitor for unexpected consequences of prolonged ANGPTL3 inhibition

  • Potential immune responses to fully human antibodies with chronic administration

  • Unknown effects of sustained reduction in HDL-C levels

• Target Population Definition:

  • Identifying patients most likely to benefit from ANGPTL3 inhibition

  • Determining optimal positioning relative to existing therapies

  • Potential for personalized approaches based on genetic testing for ANGPTL3 variants

  • Balancing cost and benefit in different patient populations

• Administration and Adherence:

  • Parenteral administration (subcutaneous injections) may affect patient acceptance

  • Different dosing frequencies (Q4W vs. Q8W) have implications for adherence

  • Need for healthcare provider administration versus self-administration

  • Cold chain requirements for antibody storage and distribution

• Outcome Evidence Requirements:

  • Current efficacy data primarily based on surrogate endpoints (lipid levels)

  • Need for cardiovascular outcome trials to demonstrate clinical benefit

  • Long duration and high cost of cardiovascular outcome studies

  • Regulatory requirements for demonstrating risk reduction beyond lipid effects

• Cost-effectiveness Considerations:

  • Biologics typically associated with higher costs than small-molecule therapies

  • Need to demonstrate value proposition compared to established treatments

  • Potential for cost-effectiveness in specific high-risk populations

  • Reimbursement challenges in different healthcare systems

Addressing these challenges requires continued research, including long-term extension studies, cardiovascular outcome trials, and real-world effectiveness studies to fully establish the role of ANGPTL3 antibodies in clinical practice and identify the patient populations who would derive the greatest benefit.